JPH0150399B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention provides a method for electrodialyzing the solution remaining after separation of the enzyme from the enzymatic racemic resolution of N-acetyl-DL-aminocarboxylic acids in the presence of L-amino acid acylase. Concerning how to post-process.

Prior Art During the synthesis of enantiomerically pure L-amino acids, the racemate obtained is often first acetylated and then the racemic N-acetyl-DL-
A bypass consists of splitting the amino acids by L-amino acid acylase. After separation of the enzyme, the L-amino acids liberated by this hydrolysis are isolated in a suitable manner. The unhydrolyzed N-acetyl-D(L)-amino acids can be provided fresh for racemic resolution after racemization.

The solution remaining after separation of the enzyme from enzymatic racemic resolution contains, for example, 0.15-0.7 mol/L-amino acid, N-acetyl derivatives (mainly N-acetyl-D-amino acids and small amounts of N-acetyl-L
-amino acid) 0.15 to 0.9 mol/and acetic acid 0.15 to
0.7 cells/(each in the form of an alkali metal salt, preferably in the form of a sodium salt) and a small amount of active salt, such as CoCl ₂ .

This solution is usually post-desalted by applying it onto an ion exchange column packed with a strongly acidic cation exchanger. Depending on the working method, a highly desalted amino acid solution, a highly acidic fraction containing anions as free acids and even a significant amount of exchanged cations, should generally be rejected as wastewater. A spent regeneration solution is obtained. An additional problem presents itself with poorly or only slightly soluble amino acids, where they may precipitate as free acids into the ion exchanger column, potentially clogging the column. This is generally addressed by increasing the temperature, but this can be a further drawback in the presence of thermally unstable compounds.

From West German Patent Application No. 2907450,
Methods for working up such solutions by electrodialysis are known. This known method is carried out in an electrodialysis apparatus consisting of a number of chambers which are alternately separated from each other by cation- and anion-exchanger membranes and which are arranged between individual pairs of electrodes. Ru. Apart from the anode and cathode cells, these chambers are constantly alternated with one supplying and diluting solution.
chamber and an adjacent chamber serves as a concentrate chamber. In this feed and dilution chamber, the solution to be worked up is rotated, and during electrodialysis the anions and cations contained in the solution permeate through the anion exchanger membrane or the cation exchanger membrane. can enter adjacent concentrate chambers.
Amphoteric amino acids are retained in the feed and dilution chambers by operating the electrodialyzer at high current densities close to the limiting current density. At this time, a PH-box (PH-Schrank) is formed in this membrane, which prevents amphoteric amino acids from passing through the membrane. In this known method, the enantiomerically pure amino acid can flow out of the dilute chamber in the form of its aqueous solution in good yields, but the anions and cations originally contained are mixed again and as a result However, it has the disadvantage that only the salt aqueous solution can flow out from the concentrate chamber.

Means for Accomplishing the Invention The method according to the invention has the following features: The electrodialysis apparatus used consists of a number of three-chamber packages, each consisting of a feed and dilute compartment 1, a concentrate compartment located on the cathode side. 2 and a concentrated liquid chamber 3 located on the anode side.
In this case, each three-chamber package receives water from both adjacent packages as OH ^- - ions.
are separated by a device 4 for decomposition into H ⁺ - ions, and after the end of the electrodialysis, an aqueous solution in the form of L-amino acids and their salts with up to 50% of the anions initially present is discharged from the diluent chamber 1; An aqueous solution of cations as hydroxides initially present is discharged from the concentrate chamber 2 located on the cathode side, and N-acetyl-D(L)-amino acid and Drain an aqueous solution of the free acid form of acetic acid.

The device 4 for water splitting contemplated in the method of the invention comprises:
It may consist of an additional chamber filled with water containing a conductive additive which does not permeate the membrane separating the chambers. Suitable conductive auxiliaries are suspended inert conductive substances such as activated carbon, carbon black or spherical metal particles or high molecular weight acids such as polyacrylic acid or polystyrene sulphonic acid or bases such as substituted or unsubstituted polyethyleneimine. It is.

The water splitting device 4 may also consist of a so-called bipolar membrane. It consists of one cation- and anion-exchanger membrane each separated from one another by a thin neutral hydrophilic layer. The structure may consist of a homogeneous block polymer or three sheets of polymer glued one above the other. Although similar equipment is readily available to those skilled in the art,
They are all based on the same basic idea: the decomposition of water molecules within an electric field and the replenishment of the adjacent concentrate chambers 2, 3 with the necessary ions.

In the electrodialysis apparatus to be used in the method of the invention,
Each feed and dilute chamber 1 comprises two different concentrate chambers, in which the initially present cations (which are OH ^- - arising from adjacent equipment 4 for
Only the anions initially present (replenished by ions to form hydroxide) can move, and in the concentrate chamber 3 located on the adjacent anode side, the initially present anions (which are replenished by the other side for water splitting) can move. (replenished by H ⁺ − ions generated from the adjacent device 4 to become free acid)
It is only possible to move.

Furthermore, the electrodialysis apparatus to be used in the method according to the invention is usually constructed as follows: a three-chamber package and the respective water-splitting device 4 for separating them are arranged between one pair of electrodes. has been done. All individual chambers are delimited by anion and cation exchanger membranes and a sealing frame, optionally with support grids. On the anode side of the electrodialysis device there is first an anode chamber delimited by a cation exchanger membrane. All subsequent rooms are
In each case, anion exchanger membranes and cation exchanger membranes are incorporated in regular alternation.
In the case of the use of a so-called bipolar membrane as device 4 for water splitting, this is installed in such a way that the anion exchange side and the cation exchange side are freely adapted to their variations. The concentrate chamber 2 of the last three-chamber package arranged on the cathode side simultaneously serves as cathode chamber.

The advantage of the process according to the invention is, inter alia, that after the end of the electrodialysis, three aqueous solutions of different composition can be discharged. This considerably expands the space for other processing. Feed as a first solution - and a dilute solution - from chamber 1 which actually still contains only L-amino acids, or at least has been sufficiently desalted so that after concentration pure L-amino acids can crystallize out. The aqueous solution is drained out. The mother liquor remaining after separation of the crystallized L-amino acids can be recycled and subjected to electrolysis again together with the fresh solution from the racemate resolution. There is therefore virtually no waste water to be removed here. As a second solution, a solution of the cations initially present in their hydroxide form can flow out of the concentrate chamber 2 arranged on the cathode side. This solution can also be completely recycled and serves to dissolve the new N-acetyl-DL-amino acid and to adjust the PH value required for enzymatic degradation. Again, no wastewater is actually produced to be removed. Finally, as a third solution, an aqueous solution of N-acetyl-D(L)-amino acid and acetic acid can be flowed out from the concentrated liquid chamber 3 arranged on the anode side, which contains ions other than H ⁺ -ions. Contains no cations,
After distilling off the acetic acid and, if necessary, the water, it can therefore be reracemized in any way and likewise completely recycled. Optionally, this third solution can also be used to obtain the D-enantiomer.

Therefore, the process of the invention is based on the waste water to be rejected, which occurs in any case in small quantities, and on the basis of its mild operating conditions (the temperature in the electrodialysis machine does not rise above 30°C), the crystallized L It is clearly superior to all known work-up methods for solutions from racemic resolution due to the achievable high purity of the amino acids and the minimal loss of material in any case. this is,
Enantiomerically pure neutral and basic L-amino acids such as lysine, tryptophan, histidine,
Suitable for obtaining phenylalanine, leucine, isoleucine, threonine, methionine, valine or arginine.

The attached FIGS. 1 and 2 schematically show two embodiments of an electrodialysis apparatus to be used in the process according to the invention: FIG.
Figure 2 shows an arrangement in which additional chambers are present between the chamber packages. 1 is the supply and dilute solution chamber, 2 is the (basic) concentrate chamber placed on the cathode side thereof, and 3 is the (acidic) concentrate chamber placed on the anode side of the supply and dilute solution chamber. 4 is an additional chamber for water splitting, K is a cation exchanger membrane, a is an anion exchanger membrane, As is an amino acid, MX is a salt, MOH
is the hydroxide produced from the cation M ⁺ and XH is the acid produced from the anion X ^- .

FIG. 2 shows an arrangement in which a so-called bipolar membrane (bM) as a device 4 for water splitting is present between each three-compartment package. 4 is therefore a bipolar membrane, and all other numbers and abbreviations have the same meaning as in FIG.

In order to carry out the method of the invention in practice, water containing the conductive auxiliary is introduced into an additional chamber which serves as a device 4 for water splitting, provided that a device of the type shown in FIG. 1 is used. do. If a device of the type shown in FIG. 2 is used, this means is omitted. Fill the anode chamber with 1N H ₂ SO ₄ . The supply and dilution chambers 1 and the concentrate chambers 2 and 3 are combined into three mutually independent pump circulation systems each having one buffer vessel. The solution to be post-treated from the racemate separation is circulated by a pump in the pump circulation system for the dilute solution chamber 1, and the solution to be post-treated from the racemate separation is circulated by a pump in the pump circulation system for the concentrate chamber 2 disposed on the cathode side for the operation of the apparatus. Water with a small amount of caustic soda and a small amount of acetic acid for operation is pumped into the device in a pump circulation system for the concentrate compartment 3 located on the anode side. Within all three pump circulation systems, the respective containing solutions are generally pumped at a linear flow rate of about 0.5 to 10 cm/sec. The device operates with direct current, the current density of which depends on the particular concentration situation and is generally advantageously between 10 and 40 mA/cm ² .

In the pump circulation system for supply and diluted liquid chamber 1, there are 4
It is advantageous to maintain a pH value of ~8, preferably from 5 to 7, which can be adjusted if necessary by adding acetic acid or caustic soda. In the pump circulation system for concentrate chamber 2 located on the cathode side, this is the PH
For the protection of the anion exchanger membrane, it may be advantageous to place the value at the upper limit of what is still tolerable in the anion exchanger membrane. The highest possible PH value depends on the type and properties of the anion exchanger membrane used. This can be maintained by at least partially neutralizing the resulting lauge by addition of solid N-acetyl-DL-amino acids. In this case, not pure mother liquor is discharged from the circulation system, but a mixture of lye and the corresponding salt of the N-acetyl-DL-amino acid. This lye should immediately be available for dissolving the new N-acetyl-DL-amino acid, so that this mixture can also be completely recycled without any problems.

When the desired degree of desalination is reached in the pump circulation system for feed and dilute chamber 1, the pump and flow are stopped. The three circulating solutions are taken off separately and further processed in the manner described above.

In continuous operation, it is possible to remove desalinated solution from the pump circulation system for the feed and dilution chamber 1 in such an amount that this circulation system is still effective. Subsequently, the difference in quantity is again compensated for by addition of undesalted decomposition solution. Correspondingly, it can also be carried out in the circulation system for the concentrate compartments 2 and 3. However, continuous feed-and-bleed-operations
Arbeitweise) is also possible.

Example The invention is illustrated in the following example.

Example 1 An electrodialysis apparatus of the type shown in FIG. 1 was used. A pump circulation system is installed, which is guided by three mutually independent buffer containers, in each case connected in parallel with the supply and dilution chambers (dilution circulation system), and the cathode side. The concentrate chamber (basic concentrate circulation system) located on the anode side and the concentrate chamber (acidic concentrate circulation system) located on the anode side are grouped together.

An additional chamber serving as a water splitting device was charged with a 2% by weight suspension of conductive carbon black in water and the anode chamber was filled with 1NH ₂ SO ₄ .

The cation exchanger membrane used was one based on polystyrene sulfonic acid and the ion exchanger membrane used was one with derivatized pyridinium end groups.

A solution of 32 g of L-methionine / 92 g of N-acetyl-D(L)-methionine as the sodium salt / 38 g of sodium acetate was added to the basic concentrate circuit with a small amount of caustic soda at startup. and the acid concentrate circulation system was charged with water with a small amount of acetic acid at startup.

Three pumps were activated and adjusted to a linear flow rate of approximately 1 cm/sec. A direct current of i=25 mA/cm ² was applied, and electrodialysis was performed for a period of time until an amount of electricity of 0.86 Faraday per mole of anion contained in the dilute solution circulation system initially flowed.

A solution with the following composition was present in the dilute circulation system: 41 g of L-methionine/36 g of N-acetyl-D(L)-methionine as the sodium salt/2 g of sodium acetate.

This solution had a PH value of about 4.9 and a temperature of about 30°C. The amount has decreased to 78% of the initial amount.

In the basic concentrate circulation system, the resulting alkaline liquid is constantly partially neutralized by addition of solid N-acetyl-DL-methionine in order to prevent an increase in pH values of more than 8, which would possibly harm the anion exchanger membrane. Upon mixing, a solution containing approximately 115 g of the sodium salt of N-acetyl-DL-methionine was obtained along with a small amount of caustic soda.

In the acidic concentrate circulation system, after the end of electrodialysis, a solution with the following composition was present: N-acetyl-D(L)-methionine as free acid.
130g/38g acetic acid/This solution had a PH value of 1.95. The current efficiency for anion transfer was approximately 90%.

The solution from the dilute circulation system continues to be about 45% of its volume.
It was evaporated and concentrated. Upon cooling, L-methionine crystallized and was centrifuged and dried. The centrifuged product has a purity of 99.7%,
[α] ²⁰ _D = 24° specific rotation, no measurable amounts of sulfate ash, 0.1% N-acetyl-D(L)-methionine and D-methionine as an impurity.
Contained only 0.2%. Recirculating the crystal mother liquor,
It was subjected to electrodialysis again with the fresh solution from the racemate resolution.

The solution from the basic concentrate circulation system was recycled to the enzymatic racemate resolution.

The solution from the acidic concentrate circuit was evaporated to almost dryness under reduced pressure. At this time, a melt-solidified product of N-acetyl-D(L)-methionine with about 0.5% residual acetic acid remained. After subsequent reracemization, this product could also be recycled to the enzymatic racemate resolution.

Example 2 The same electrodialysis equipment as in Example 1 was used.

The water-splitting chamber was charged with a solution of 15% by weight polyacrylic acid in water. Residual membrane laminate as a membrane separating the chambers
The same anions and cations used in
An exchanger membrane was used.

Desalting of the same solution as in Example 1, using two diluent chambers and a total of four concentrate chambers, under otherwise identical conditions, provided the same results at an average cell voltage of about 40V. Loss of polyacrylic acid into the concentrate chamber could not be proven.

Example 3 In the same membrane stack as in the previous example, the water splitting chamber was replaced by a bipolar membrane. The stack contained three dilute compartments, a total of six concentrate compartments, and two electrode compartments.

A solution of the following composition: L-valine 35 g/N-acetyl-D(L)-valine as sodium salt 54 g/ and sodium acetate 11 g/ is charged into the dilute liquid circulation system, and a concentrated solution similar to that in Example 1 is added. Start-up solution suitable for the circulation system and electrode circulation system
Filled with 0.5M _{Na2SO4 -} solution. After starting the pump, a direct current with a current density i=25 mA/cm ² was applied to the cell, adapting to an average cell voltage of 30 V. Anion 1 initially contained in the dilute liquid circulation system
After passing a quantity of electricity of 0.92 Huaraday per cell,
The desalted solution had the following composition: L-valine 41 g/ N-acetyl-D(L)-valine as sodium salt 22 g/ and sodium acetate 0 g/ The solution had a PH value of approximately 5. , its temperature is about 30
It was warm at ℃.

In the acidic concentrate circulation system the following composition: N-acetyl-D(L)-valine as free acid
A solution of 130g/ and acetic acid of 86g/ was able to flow out.

The current efficiency for anion transfer was about 75%.

Evaporate and concentrate the solution from the dilute circulation to approximately
40% and cooled. L-valine could be isolated by centrifugation with a crystal yield of 60%.

The crystalline product had a content of 99%, an optical rotation of [α] ²⁵ _D =27.4° and an ash content of 0.01%.

The acidic concentrate solution was recycled to the racemic resolution as in Example 1.

[Brief explanation of drawings]

FIG. 1 shows one embodiment of an electrodialysis apparatus used to carry out the method of the invention, and FIG. 2 shows another embodiment. 1... Supply and dilute liquid chamber, 2... Basic concentrate chamber, 3... Acidic concentrate chamber, 4... Water decomposition chamber, K...
...Cation exchanger membrane, a...Anion exchanger membrane,
As...amino acid, MX...salt, MOH...hydroxide generated from cation M ⁺ , XH...anion X ^-
Acid generated from bM...Bipolar membrane.

[Claims]

1. A reagent for quantifying hydrogen peroxide, comprising a compound portion having a residue obtained by removing one hydrogen atom from active methylene or active methine, and a fluorescent portion. 2. A hydrogen peroxide quantitative reagent consisting of a compound moiety having a residue from which one hydrogen atom has been removed from active methylene or active methine and a fluorescent moiety, a hydrogen donor, and peroxidase is added to a sample containing hydrogen peroxide. 1. A method for quantifying hydrogen peroxide, which comprises causing hydrogen peroxide to react and measuring the fluorescence intensity of the resulting fluorescent substance.

Claims (1)

How to post-process the liquid. 2. The method according to claim 1, wherein the device 4 for water splitting consists of an additional chamber filled with water containing a conductive auxiliary agent which does not permeate through the membrane delimiting the chamber. . 3. The method of claim 2, wherein the conductive aid is a suspended inert conductive material. 4. The method according to claim 3, wherein the conductive auxiliary agent is a high molecular weight acid or base. 5. The method according to claim 1, wherein the water splitting device 4 comprises a so-called bipolar membrane.